In medical checkups, blood tests are widely conducted for the purpose of health condition check and early detection of an illness. In order to analyze many samples for many kinds of targets in test menu, samples are accumulated at a general hospital or testing laboratory where blood tests are conducted using a large clinical chemistry analyzer with a high throughput. Therefore, considering the time for transporting samples, waiting time for testing, time for making summary of testing and time for delivering summaries, usually it takes several days from sampling until testing results become available. In the case of medical checkups, in most cases this time lag does not matter since many people have a medical checkup every six months or once a year. However, in so-called point of care testing (POCT) where test results should be available on the site of sampling such as emergency tests including intraoperative tests, tests on ambulant patients, tests in ambulances, tests at clinics, and self-examinations at home including self-monitoring of blood glucose, the need for immediate availability of test results cannot be met if a clinical chemistry analyzer as mentioned above is used. In addition, since a clinical chemistry analyzer is expensive and must be operated by a specialist, it is not realistic to introduce a clinical chemistry analyzer at each site. The required device for POCT should be inexpensive enough for each site to afford to introduce, be compact enough to be carried to each site and be operable by a non-specialist user even though it may be not up to a clinical chemistry analyzer in terms of the number of targets in test menu and throughput.
The targets in test menu which the testing device for POCT is required to handle are as follows: sodium, potassium, and chloride as blood electrolytes which are measured in conventional blood tests, oxygen partial pressure, carbon dioxide partial pressure, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, urea nitrogen, lactase dehydrogenase (LD), gamma glutamyltransferase, cholesterol, bilirubin, choline esterase, neutral lipid, glucose, hematocrit and so on. The targets except electrolytes and hematocrit among them are usually measured using chemical reactions such as enzyme reactions. Measurement methods based on enzyme reactions which are used by currently commercially available testing devices for POCT are classified into a colorimetric reaction method and an amperometric assay (using enzyme electrodes). Since the colorimetric reaction method is also used in clinical chemistry analyzers, it is relatively easy to develop a POCT device which employs this measurement method. However, since an optics system is needed for colorimetric reactions, the size of a POCT device which can handle many kinds of targets should be a desk-top size or so. On the other hand, the amperometric assay is an electrical measurement method which does not require an optics system, so a device which adopts this method can be small or palm-sized even when it can measure many kinds of targets.
In the amperometric assay, three electrodes, which are a working electrode of gold or platinum, a counter electrode, and a reference electrode for keeping the working electrode potential constant, are placed in a solution in which an enzyme and redox compounds coexist. The working electrode, counter electrode, and reference electrode are connected to an amperometric device such as a potentiostat so as to enable measurement of current values which change when a voltage is applied between the working electrode and counter electrode. When a sample (for example, blood) containing a target analyte is added to the solution, the oxidization of target analyte is catalyzed by the enzyme and at the same time the redox compound in an oxidized state is brought into a reduced state. When a given voltage which can oxidize the redox compound is applied to the working electrode, the redox compound in a reduced state is oxidized on the working electrode, causing an electric current to flow in the working electrode. The oxidized redox compound reacts with the target analyte again by the enzyme catalysis and enters a reduced state. As this reaction is repeated, the oxidation reaction of the target analyte by the enzyme can be detected as an electric current. At this time, a redox compound with a sufficient concentration and a sufficiently large working electrode are needed so that a current value depending on the concentration of the target analyte is obtained, namely in the reaction system the target analyte concentration limits the reaction rate. Also, in order to minimize the voltage drop due to the electric resistance of the solution, it is desirable that the counter electrode and working electrode be as near to each other as possible. Furthermore, in order to simplify the constitution, one electrode which functions as both the reference electrode and counter electrode may be used and in that case, in order to minimize the voltage loss in the counter electrode, it is desirable that the counter electrode be as large as possible.
Since the sensitivity which is required for the glucose sensor for measuring the blood glucose level is not so high, the blood glucose level can be measured from a few drops of blood (for example, Patent Document 1). However, in a testing device for POCT which handles ordinary test menu, a larger volume of blood is required in order to maintain the sensitivity. For example, the i-Stat (Non-patent Document 1) developed as a testing device for POCT requires about 65 μl of blood. Although the required volume of blood can be decreased by decreasing the electrode area, in the amperometric assay, simply decreasing the electrode area leads to a decrease in signal (namely, current value), so there is difficulty in decreasing the electrode area.
A potentiometric assay is known as an electric measurement method in which signals do not depend on the electrode area. The potentiometric assay includes a measurement electrode (working electrode) made of gold or platinum and a reference electrode and an enzyme and a redox compound exist in a measuring solution (Patent Document 2). The measurement electrode and reference electrode are connected to a device which measures a voltage, such as a voltmeter. As a target analyte is added to a measuring solution, the target analyte is oxidized by enzyme reaction and at the same time the redox compound in an oxidized state is brought into a reduced state. The surface potential of the measurement electrode which is generated at that time is calculated in accordance with the Nernst Equation given below:
                    E        =                              E            0                    +                                    RT                              n                ⁢                                                                  ⁢                F                                      ⁢                          ln              ⁡                              (                                                      C                    ox                                    /                                      C                    red                                                  )                                                                        [                  Equation          ⁢                                          ⁢          1                ]                E: Surface potential of a measurement electrode    E0: Standard potential of a redox compound    R: Gas constant    T: Absolute temperature    n: Charge difference between the oxidized form and reduced form of the redox compound    F: Faraday constant    Cox: Concentration of the oxidized form of the redox compound    Cred: Concentration of the reduced form of the redox compound
The above equation does not include the electrode area and the surface potential does not depend on the electrode area. Therefore, it is possible to decrease the electrode area without a decrease in signal intensity and reduce the required volume of sample. In addition, the distance between the reference electrode and measurement electrode does not pose the problem of voltage drop as seen in the amperometric assay and does not affect the measuring accuracy.
The amperometric assay and the potentiometric assay differ not only in the dependence of signals on the electrode area but also in whether signals depend on the reaction rate or reaction volume of enzyme reaction. Specifically, while signals proportional to the enzyme reaction rate are obtained in the amperometric assay, signals which depend on the volume of reduced substance (or oxidized substance) produced by enzyme reaction are obtained in the potentiometric assay. For this reason, while only the rate assay in which the reaction rate of enzyme reaction is measured can be used in the amperometric assay, the end point assay in which the total volume of reaction product is measured can be used in addition to the rate assay in the potentiometric assay. As a characteristic of an enzyme, its reaction rate is proportional to its concentration in a low substrate concentration range, but when the substrate concentration is high, the reaction rate is no longer proportional to the substrate concentration and when the concentration further goes up, the reaction rate becomes constant. This threshold concentration is called Michaelis constant and when the target analyte concentration in blood is not less than the Michaelis constant, the sample must be diluted for measurement in the rate assay, making measurement operation complicated. On the other hand, in the end point assay, since the total volume of reaction product is measured, there is no such a limitation and measurement can be made without dilution. One of enzymes of this type is cholesterol dehydrogenase and theoretically the potentiomeric assay may be said to be a method which can measure cholesterols without dilution.